Proteases of the endosomal/lysosomal system play important roles in a number of physiological and pathological processes, including the immune response, arthritis, bone remodeling and tumor growth. Although the enzymes are primarily lysosomal, they have been detected in endosomes, on the plasmamembrane and in extracellular fluids. The origin and activity of the non- lysosomal forms of the enzymes are not clear. The long-term objectives of this project are to develop procedures that enable the active enzymes to be identified so that they can be selectively inhibited to control their activity in pathological and physiological processes. A new method has been developed using an active-site directed, covalently binding inhibitor to target intracellular compartments. This technique has been used in one cell culture system to show that active proteases are found in both endosomes and lysosomes, but are not secreted or membrane bound. A new series of reagents will be developed to enable evaluation of the unversality of this observation. The specific aims are: 1. To determine whether newly synthesized lysosomal cysteine proteases are preferentially delivered to, and activated in endosomes. Biosynthetic pulse-chase experiments will be used in concert with a transferrin-conjugated covalently reacting peptidyl inhibitor to identify newly synthesized proteases. 2. To determine whether delivery of endocytosed proteins to protease-positive compartments occurs similarly in different cell types. A range of cell types will be examined using a protein conjugated inhibitor to identify active proteases in cells. 3. To determine whether molecules taken up by different endocytic processes are delivered to similar protease- positive compartments. A conjugation system will be developed to permit inhibitors and targeting proteins to be interchanged using biotin-avidin chemistry. These studies will show which molecular forms of the lysosomal proteases are capable of degrading endocytosed proteins, enabling the generation of more specific reagents to regulate endosomal proteolysis.